Research Article Immunomodulatory and Antidiabetic Effects...

9
Research Article Immunomodulatory and Antidiabetic Effects of a New Herbal Preparation (HemoHIM) on Streptozotocin-Induced Diabetic Mice Jong-Jin Kim, 1 Jina Choi, 1 Mi-Kyung Lee, 2 Kyung-Yun Kang, 3 Man-Jeong Paik, 3 Sung-Kee Jo, 4 Uhee Jung, 4 Hae-Ran Park, 4 and Sung-Tae Yee 1,3 1 Department of Biology, Sunchon National University, 255 Joongang-Ro, Seokhyeon-Dong, Suncheon 549-742, Republic of Korea 2 Department of Food and Nutrition, Sunchon National University, Suncheon, Republic of Korea 3 Department of Pharmacy, Sunchon National University, 255 Joongang-Ro, Seokhyeon-Dong, Suncheon 549-742, Republic of Korea 4 Radiation Research Division for Bio-Technology, Advanced Radiation Technology Institute, Jeongeup Campus of Korea Atomic Energy Research Institute (KAERI), Jeongeup, Republic of Korea Correspondence should be addressed to Sung-Tae Yee; [email protected] Received 12 March 2014; Revised 9 May 2014; Accepted 31 May 2014; Published 18 June 2014 Academic Editor: Cheng-Rui Li Copyright © 2014 Jong-Jin Kim et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HemoHIM (a new herbal preparation of three edible herbs: Angelica gigas Nakai, Cnidium officinale Makino, and Paeonia japonica Miyabe) was developed to protect immune, hematopoietic, and self-renewal tissues against radiation. is study determined whether or not HemoHIM could alter hyperglycemia and the immune response in diabetic mice. Both nondiabetic and diabetic mice were orally administered HemoHIM (100mg/kg) once a day for 4 weeks. Diabetes was induced by single injection of streptozotocin (STZ, 200mg/kg, i.p.). In diabetic mice, HemoHIM effectively improved hyperglycemia and glucose tolerance compared to the diabetic control group as well as elevated plasma insulin levels with preservation of insulin staining in pancreatic -cells. HemoHIM treatment restored thymus weight, white blood cells, lymphocyte numbers, and splenic lymphocyte populations (CD4 + T and CD8 + T), which were reduced in diabetic mice, as well as IFN- production in response to Con A stimulation. ese results indicate that HemoHIM may have potential as a glucose-lowering and immunomodulatory agent by enhancing the immune function of pancreatic -cells in STZ-induced diabetic mice. 1. Introduction Diabetes mellitus (DM) is one of the leading causes of morbidity and mortality worldwide [1]. Increases in the aging population, consumption of calorie-rich diets, obesity, and sedentary lifestyle have led to a tremendous surge in the number of diabetics [2]. Likewise, incidence of diabetes in Korea has increased rapidly in the past 10 years, becoming the 4th leading cause of death [3]. Type 1 DM results from the selective destruction of insulin-producing -cells in pancreatic islets, and it is primar- ily a T cell-mediated autoimmune disease directed against one or more -cell autoantigens [4, 5]. is state is char- acterized by limited weight gain, polyuria, polydipsia, and polyphagia attributed to the decreased capability of insulin to stimulate glucose uptake and utilization in target tissues due to insulin resistance, insulin insufficiency, and changes in other factors such as glucagon, thyroxin, glucocorticoid, and catecholamines [1]. A number of studies on oral antihyperglycemic agents derived from plants used in traditional Oriental medicine have been conducted, and many of the plants were found to have good activity [6]. e World Health Organization (WHO) has also recommended the evaluation of plants’ effectiveness whenever safe modern drugs are unavailable [7]. is has led to an increased demand for research on natural products with antidiabetic activity as well as mini- mal to no side effects [8]. Unfortunately, complete therapy for DM and its complications has not been established yet. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2014, Article ID 461685, 8 pages http://dx.doi.org/10.1155/2014/461685

Transcript of Research Article Immunomodulatory and Antidiabetic Effects...

Page 1: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

Research ArticleImmunomodulatory and Antidiabetic Effects of a NewHerbal Preparation (HemoHIM) on Streptozotocin-InducedDiabetic Mice

Jong-Jin Kim,1 Jina Choi,1 Mi-Kyung Lee,2 Kyung-Yun Kang,3 Man-Jeong Paik,3

Sung-Kee Jo,4 Uhee Jung,4 Hae-Ran Park,4 and Sung-Tae Yee1,3

1 Department of Biology, Sunchon National University, 255 Joongang-Ro, Seokhyeon-Dong, Suncheon 549-742, Republic of Korea2Department of Food and Nutrition, Sunchon National University, Suncheon, Republic of Korea3 Department of Pharmacy, Sunchon National University, 255 Joongang-Ro, Seokhyeon-Dong, Suncheon 549-742, Republic of Korea4Radiation Research Division for Bio-Technology, Advanced Radiation Technology Institute, Jeongeup Campus of KoreaAtomic Energy Research Institute (KAERI), Jeongeup, Republic of Korea

Correspondence should be addressed to Sung-Tae Yee; [email protected]

Received 12 March 2014; Revised 9 May 2014; Accepted 31 May 2014; Published 18 June 2014

Academic Editor: Cheng-Rui Li

Copyright © 2014 Jong-Jin Kim et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HemoHIM (a new herbal preparation of three edible herbs: Angelica gigasNakai, Cnidium officinaleMakino, and Paeonia japonicaMiyabe) was developed to protect immune, hematopoietic, and self-renewal tissues against radiation. This study determinedwhether or not HemoHIM could alter hyperglycemia and the immune response in diabetic mice. Both nondiabetic and diabeticmice were orally administered HemoHIM (100mg/kg) once a day for 4 weeks. Diabetes was induced by single injection ofstreptozotocin (STZ, 200mg/kg, i.p.). In diabetic mice, HemoHIM effectively improved hyperglycemia and glucose tolerancecompared to the diabetic control group as well as elevated plasma insulin levels with preservation of insulin staining in pancreatic𝛽-cells. HemoHIM treatment restored thymus weight, white blood cells, lymphocyte numbers, and splenic lymphocyte populations(CD4+ T and CD8+ T), which were reduced in diabetic mice, as well as IFN-𝛾 production in response to Con A stimulation.Theseresults indicate that HemoHIMmay have potential as a glucose-lowering and immunomodulatory agent by enhancing the immunefunction of pancreatic 𝛽-cells in STZ-induced diabetic mice.

1. Introduction

Diabetes mellitus (DM) is one of the leading causes ofmorbidity andmortality worldwide [1]. Increases in the agingpopulation, consumption of calorie-rich diets, obesity, andsedentary lifestyle have led to a tremendous surge in thenumber of diabetics [2]. Likewise, incidence of diabetes inKorea has increased rapidly in the past 10 years, becomingthe 4th leading cause of death [3].

Type 1 DM results from the selective destruction ofinsulin-producing𝛽-cells in pancreatic islets, and it is primar-ily a T cell-mediated autoimmune disease directed againstone or more 𝛽-cell autoantigens [4, 5]. This state is char-acterized by limited weight gain, polyuria, polydipsia, andpolyphagia attributed to the decreased capability of insulin

to stimulate glucose uptake and utilization in target tissuesdue to insulin resistance, insulin insufficiency, and changes inother factors such as glucagon, thyroxin, glucocorticoid, andcatecholamines [1].

A number of studies on oral antihyperglycemic agentsderived from plants used in traditional Oriental medicinehave been conducted, and many of the plants were foundto have good activity [6]. The World Health Organization(WHO) has also recommended the evaluation of plants’effectiveness whenever safe modern drugs are unavailable[7]. This has led to an increased demand for research onnatural products with antidiabetic activity as well as mini-mal to no side effects [8]. Unfortunately, complete therapyfor DM and its complications has not been establishedyet.

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014, Article ID 461685, 8 pageshttp://dx.doi.org/10.1155/2014/461685

Page 2: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

2 Evidence-Based Complementary and Alternative Medicine

In traditional Oriental medicine, many herbs or herbalprescriptions comprising several medicinal plants have beenreputed to promote health, improve the defense mechanismsof the body, and enhance longevity [9]. A newherbal prepara-tion (HemoHIM) was designed to protect self-renewing tis-sues as well as promote recovery of the immune system [10].HemoHIM was first prepared by adding its polysaccharidefraction to the hotwater extract of a herbalmixture consistingof Angelica Radix, Cnidii Rhizoma, and Paeonia Radix [11].In our previous study, HemoHIM rescued white blood cells,and lymphocytes were reduced using ionizing radiation (IR).Also,we previously observed thatHemoHIMwas effective forthe restoration of impaired immune functions [9] and its anti-inflammatory activity against carrageenan-induced edema,the formation of granulation tissues by cotton pellet, andexperimental colitis by 2,4,6-trinitrobenzene sulfonic acid[11]. In addition,HemoHIMenhances the therapeutic efficacyin tumor-bearingmice treatedwith a chemotherapeutic agentor IR [12, 13]. Overall, these data suggest that HemoHIMcan act as a potential modifier of biological responses. Thegeneral composition of HemoHIM is 60.4% carbohydrate,6% protein, and 33.6% other including polyphenols (data notshown).The immunemodulating components in HemoHIMwere the ethanol-insoluble fraction [14, 15], the polysaccha-ride content of which was 40.9% (±3.8) (data not shown).Other carbohydrate components in HemoHIM were acidic-polysaccharide ethanol-soluble fraction, the polysaccharidecontent of which was 19.5% (data not shown). In addition, thefunctional components included in the ethanol-soluble frac-tion of HemoHIM were gallic acid [0.2% (±0.06)], chloro-genic acid [0.33% (±0.05)], paeoniflorin [1.32% (±0.15)],nodakenin [0.58% (±0.04)], and benzoic acid [0.17% (±0.05)].In particular, these herbs are listed as raw materials in theKorean Food Code. Finally, HemoHIM has been proven tobe safe for long-term administration [16, 17].

Streptozotocin (STZ) is 1-methyl-1-nitrosourea derivativeof 2-deoxy-Dglucose and a broad antibacterial agent thatcan cause symptoms of diabetes. Various studies reportedantidiabetic effects using STZ-induced diabetic mice model[18, 19]. In the current study, we investigated whether or notHemoHIM could improve immune function and antidiabeticeffect on STZ-induced diabetic mice.

2. Materials and Methods

2.1. Preparation of HemoHIM. A mixture of three ediblemedicinal herbs, Angelica Radix (root of Angelica gigasNakai), Cnidii Rhizoma (rhizome of Cnidium officinaleMakino), and Paeonia Radix (root of Paeonia japonicaMiyabe) was decocted for 4 h in boiling water to obtain atotal extract. A part of the total extract was fractionated intoan ethanol-soluble fraction and an ethanol-insoluble polysac-charide fraction by precipitation in 80% ethanol. HemoHIMwas prepared by adding the polysaccharide fraction to theother part of the total extract [9].

2.2. Experimental Animals. Six-week-old male ICR micewere purchased from Orient Inc. (Charles River Technology,

Seoul, Korea). The mice were individually housed and main-tained at a controlled temperature (22 ± 2∘C) and relativehumidity (50±5%)under a 12 h light-dark cycle. Allmicewerefed a pelletized commercial chow diet for 7 days after arrival.Next, the animals were randomly divided into four groupsof 10 mice each: nondiabetic control group (nondiabetic),nondiabetic group administered HemoHIM (nondiabetic +HemoHIM), diabetic control group (diabetic), and diabeticgroup administered HemoHIM (diabetic + HemoHIM).The mice had access to food and water ad libitum. Bothnondiabetic and diabetic mice were orally administeredHemoHIM (100mg/kg) once a day for 4 weeks. Body weightsof mice were measured weekly. All mice were treated in strictaccordance with Sunchon National University InstitutionalAnimal Care andUse Committee (SCNU IACUC) guidelinesfor the care and use of laboratory animals. All procedureswere approved by the SCNU IACUC.

2.3. Induction of Diabetes in Mice. Diabetes was induced by asingle injection of STZ (200mg/kg body weight) (Sigma, St.Louis, MO, USA) freshly dissolved in 0.1M citrate buffer (pH4.2) into the intraperitoneal injection. Nondiabeticmice wereinjectedwith citrate buffer alone. After 48 h, only STZ-treatedmice that exhibited a fasting blood glucose level ≥200mg/dLwere used in the study.

2.4. Fasting Blood Glucose Levels and Oral Glucose ToleranceTest (OGTT). Blood glucose levels weremonitored in venousblood drawn from the tail using a glucometer (ACCU-CHEK, Roche, Germany) every week after 12 h of fasting.The OGTT was performed in the 4th week. Following 12 hof fasting, the mice were orally administered glucose at1 g/kg of body weight, after which blood glucose levels weremeasured from the tail vein at 30, 60, and 120min afterglucose administration.

2.5. Plasma Insulin Levels. Blood was collected in a heparin-coated tube and centrifuged at 3,000 rpm for 5min at 4∘C.Theplasma insulin levels were determined using a quantitativesandwich enzyme immunoassay kit (ELISA kit, ShibayagiCo., Ltd., Japan).

2.6. Immunohistochemistry of Pancreas. To measure insulinlevels, immunohistochemical analysis was performed using amouse anti-insulinmonoclonal antibody (BioGenex, Nether-lands). Briefly, paraffin sections of 4𝜇m thickness weretreated with 3% H

2O2in methanol for 30min to block any

endogenous peroxidase, followed by washing with 0.01Mphosphate buffer for 10min. Next, the sections were pro-cessed by an indirect immunoperoxidase technique using acommercial kit (ABC kit, DAKO, USA) with secondary anti-bodies. The sections were then counterstained with Mayer’shematoxylin, and the staining was visualized by incubationwith diaminobenzidine (DAB) (Zymed Laboratories, SanFrancisco, CA, USA) and counterstaining with 1% methylgreen for 1min [20].

Page 3: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

Evidence-Based Complementary and Alternative Medicine 3

Table 1: Changes in organ weights in mice administered HemoHIM.

Nondiabetic Nondiabetic + HemoHIM Diabetic Diabetic + HemoHIMLiver (mg/g) 39.25 ± 2.74 38.93 ± 0.97 48.39 ± 3.45∗ 42.07 ± 4.55#

Kidney (mg/g) 13.49 ± 0.23 13.44 ± 0.61 19.79 ± 1.01∗ 15.22 ± 1.61∗,#

Thymus (mg/g) 1.30 ± 0.12 1.14 ± 0.11 0.58 ± 0.10∗ 1.03 ± 0.14∗,#

Lung (mg/g) 4.84 ± 2.74 4.92 ± 0.10 6.06 ± 0.36∗ 5.54 ± 0.26∗,#

Spleen (mg/g) 2.73 ± 0.19 2.96 ± 0.21 2.56 ± 0.41 2.77 ± 0.44Heart (mg/g) 4.65 ± 0.22 4.74 ± 0.43 4.42 ± 0.11 4.41 ± 0.32Values are expressed as means ± S.D.∗𝑃 < 0.05 compared with nondiabetic group.

#𝑃 < 0.05 compared with diabetic group.

2.7. Plasma Hematological Change and Biomarkers. Hema-tological change analysis was carried out using an auto-matic analyzer (HEMAVET 850, USA) for white bloodcells (WBCs) and lymphocytes. Serum GOT (glutamateoxaloacetate transaminase), GPT (glutamic pyruvic transam-inase), LDH (lactate dehydrogenase), and ALP (alkalinephosphatase) activities were determined using an automaticanalyzer (FUJI DRI-CHEM 3500, Japan).

2.8. Cell SurfaceMarkers Assay. Spleen cells (1× 106 cells/mL)were blocked with anti-CD16/32 (Fc𝛾II/III receptor) mAb for30min at 4∘C and then washed with PBS solution containing1% FBS and 0.1% NaN

3. The cells were stained with PE-

conjugated anti-CD8 mAb and FITC-conjugated anti-CD4mAb or FITC-conjugated anti-CD19 mAb for 30min at4∘C. Stained cells were then washed and detected by a flowcytometer (COULTER Epics XL, USA).

2.9. Cytokines and Immunoglobulins Analysis. Spleen cells (5× 106 cells/mL) were treated with Con A (1𝜇g/mL) or LPS(10 𝜇g/mL) for 24 or 48 h, after which culture supernatantswere harvested and stored at −20∘C until use. Cytokine andimmunoglobulin contents were measured by an enzyme-linked immunosorbent assay kit (ELISA) (Pharmigen, SanDiego, CA, USA). All samples were tested in triplicate inaccordancewith standard curves.The sensitivity of each assaywas as follows: IL-2, IFN-𝛾, IL-6, and TNF-𝛼, 10 pg/mL; IgMand IgG1, 100 pg/mL.

2.10. Statistical Analysis. All data are presented as themean ±SD. Statistical analyses were performed using the SPSS pro-gram (SPSS, Chicago, IL). Student’s t-test was used to assessthe differences between the groups. The nondiabetic groupwas compared with the nondiabetic + HemoHIM, diabetic,and diabetic + HemoHIM groups. The effects of HemoHIMadministration were also compared within diabetic micegroups. Values of 𝑃 < 0.05 were considered to be statisticallysignificant.

3. Results

3.1. Body Weight and Organ Weight Changes. During theexperimental period, the body weight of diabetic mice wassignificantly lower than that of nondiabetic mice. However,

20

25

30

35

40

45

0 day 1 week 2 week 3 week 4 week

Body

wei

ght (

g)

Nondiabetic DiabeticNondiabetic + HemoHIM Diabetic + HemoHIM

∗∗

###

#

Figure 1: Effects of HemoHIM on changes in body weight of STZ-induced diabetic mice. ICR mice orally administrated HemoHIM(4 weeks) before being injected with STZ. Mice body weights weremeasured weekly. Values are expressed as the mean ± S.D. ∗𝑃 <0.05 compared with nondiabetic group. #

𝑃 < 0.05 compared withdiabetic group.

HemoHIM administration suppressed the reduction of bodyweight due to diabetes from the 1st week in diabetic mice(Figure 1).

Relative weights of the liver, kidney, and lung weresignificantly higher in diabetic control mice compared tonondiabetic control mice, whereas thymus weight was lower.Although HemoHIM administration did not affect the organweights of nondiabetic mice, it effectively recovered those ofdiabetic mice (Table 1).

3.2. Blood Glucose Level and Oral Glucose Tolerance Test(OGTT). HemoHIM administration did not affect bloodglucose levels in nondiabetic mice. However, it significantlyreduced blood glucose levels in diabetic mice treated withHemoHIM compared to the diabetic control group from

Page 4: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

4 Evidence-Based Complementary and Alternative Medicine

0

50

100

150

200

250

300

0 day 1 week 2 week 3 week 4 week

BGL

(mg/

dL)

Nondiabetic DiabeticNondiabetic + HemoHIM Diabetic + HemoHIM

∗∗

∗∗

#

##

#

(a)

0

100

200

300

400

500

600

0 min 30 min 60 min 120 min

BGL

(mg/

dL)

Nondiabetic DiabeticNondiabetic + HemoHIM Diabetic + HemoHIM

#

#

##

(b)

Figure 2: ICRmice orally administratedHemoHIM (4weeks) before being injected with STZ. Blood glucose levels weremonitored in venousblood drawn from the tail (a). The oral glucose tolerance test was performed in the 4th week. Following 12 h of fasting, the mice were orallyadministered glucose at 1 g/kg of body weight, after which blood glucose levels were measured from the tail vein at 30, 60, and 120min afterglucose administration (b). Values are expressed as means ± S.D. ∗𝑃 < 0.05 compared with nondiabetic group. #

𝑃 < 0.05 compared withdiabetic group.

the 2nd to 4th week. At the end of experiment, HemoHIMadministration had significantly reduced the fasting bloodglucose level by 26% in diabetic mice (Figure 2(a)).

To determine the effects of HemoHIM on the postpran-dial glucose level, we performed an oral glucose tolerance test.Blood glucose reached its highest level at 30min after glucoseadministration. Blood glucose levels of the diabetic grouptreated with HemoHIM were significantly lower than thoseof the diabetic control group (Figure 2(b)). Thus, HemoHIMeffectively improved fasting glucose and postprandial bloodglucose levels in STZ-induced diabetic mice.

3.3. Plasma Insulin Level and Pancreatic Immunohistochem-istry. There was no difference in the plasma insulin levelbetween nondiabetic mice and nondiabetic mice adminis-tered HemoHIM. The plasma insulin level of the diabeticcontrol group was significantly reduced compared to thatof the nondiabetic group, whereas HemoHIM significantlyincreased the plasma insulin level by approximately 2-fold(Figure 3(a)).

Reduction of the 𝛽-cell population was evident in thediabetic control group compared to the nondiabetic groupbased on changes in the immunohistochemistry of thepancreas. In the nondiabetic group, insulin-positive 𝛽-cellswere found throughout the islets, whereas only a few insulin-positive 𝛽-cells were sporadically scattered in the islets of thediabetic group.On the other hand, the insulin-positive𝛽-cells

in the diabetic group treated with HemoHIM showed strongstaining and were preserved within distinct boundaries com-pared to those of the diabetic control group (Figure 3(b)).

3.4. Splenic Lymphocyte Subpopulations. Splenic lymphocyteCD4+ T (helper T cell) and CD8+ T (cytotoxic T cell) cellpopulations were significantly reduced in diabetic controlmice compared to nondiabetic mice (Figure 4). However,HemoHIM increased the number of CD4+ and CD8+ lym-phocyte subpopulations compared to the diabetic controlgroup. Counts of CD19+ cells were not different between thegroups (Figure 4).

3.5. Production of Cytokine and Immunoglobulins by Spleno-cytes. The IFN-𝛾 level of the diabetic group was reduced toabout 56% of that of the nondiabetic group. On the otherhand, the diabetic group treated with HemoHIM showedrestored IFN-𝛾 levels. The IL-6 level increased in the diabeticgroup, showing values significantly higher than those ofthe nondiabetic group, whereas the diabetic group treatedwith HemoHIM showed restored IL-6 levels (Table 2). Afterstimulation with LPS, immunoglobulins were measured inthe supernatant of splenocytes. Levels of IgM and IgG1were increased in the diabetic mice compared with thenondiabetic group. On the other hand, the diabetic grouptreated with HemoHIM showed reduced immunoglobulinsecretion levels (Table 2).

Page 5: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

700(p

g/m

L)

Nondiabetic DiabeticNondiabetic + Diabetic +HemoHIM HemoHIM

#

(a)

Nondiabetic

Diabetic

Nondiabetic + HemoHIM

Diabetic + HemoHIM

(b)

Figure 3: ICRmice orally administratedHemoHIM (4 weeks) before being injected with STZ. Effect of HemoHIM administration on plasma(a) and pancreatic 𝛽-cell ((b), ×200) insulin levels in STZ-induced diabetic mice.Pancreas was immunohistochemically stained as describedin Materials and Methods. Values are expressed as means ± S.D. ∗𝑃 < 0.05 compared with nondiabetic group. #

𝑃 < 0.05 compared withdiabetic group.

0.0

10.0

20.0

30.0

40.0

50.0

(%)

CD4 CD8 CD19

NS

Nondiabetic DiabeticNondiabetic + HemoHIM Diabetic + HemoHIM

#

#

Figure 4: Effects of HemoHIM on splenocyte subpopulation inSTZ-induced diabetic mice. Spleen cells (1 × 106 cells/mL) werestained with PE-anti-CD8, FITC-anti-CD4, or FITC-anti-CD19 for30min at 4∘C after blocking Fc𝛾II/III receptor. Stained cells wereanalyzed by a flow cytometer. Values are expressed as means ± S.D.∗

𝑃 < 0.05 compared with nondiabetic group. #𝑃 < 0.05 compared

with diabetic group.

3.6. Blood Hematological and Plasma Biomarker Changes.Lymphocyte and WBC numbers were significantly reducedin the diabetic groups compared to nondiabetic groups.However, HemoHIM treatment recovered these numbers tooclose to nondiabetic values (Figure 5). Although serumGOT,

GPT, LDH, andALP activities were significantly higher in thediabetic group than in the nondiabetic group by 2.2-, 2.2, 1.7,and 2-fold, respectively, HemoHIM administration reducedthem compared to the diabetic control group (Table 3).

4. DiscussionThis study was conducted to investigate the effects of anew herbal preparation (HemoHIM) on immune functionand hyperglycemia in STZ-induced diabetic mice as a noveldiabetic remedy. HemoHIM consists of three kinds of edibleherbs, Angelica gigas Nakai, Cnidium officinale Makino, andPaeonia japonica Miyabe, which have been reported aspossessing antidiabetic and antihyperglycemic activities [21–23].

Since STZ is known to cause selective destruction of𝛽-cells within islets of Langerhans, resulting in markedreduction of insulin levels, it makes sense that glycogenlevels in tissues decrease as they depend on insulin for influxof glucose [24–26]. HemoHIM administration significantlyreduced fasting blood glucose levels while elevating plasmainsulin levels in STZ-induced diabetic mice. The plasmainsulin level of diabetic control mice was about 35% of thatof nondiabetic mice. However, HemoHIM administrationelevated the plasma insulin level by 200% compared tothat of the diabetic control group. In this study, OGTT ofdiabetic mice was significantly higher than that in nondia-betic mice. However, HemoHIM administration effectivelyimproved postprandial blood glucose levels. As such, theseresults suggest that constituents of HemoHIM may inhibitthe increase in blood glucose levels due to enhancementof insulin secretion in STZ-induced diabetic mice. We alsoobserved that HemoHIM effectively preserved pancreatic 𝛽-cells compared to the diabetic control group using immuno-histochemistry assay. In addition, some studies reported thatSTZ-diabetic mice induce infiltration of lymphocytes to the

Page 6: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

6 Evidence-Based Complementary and Alternative Medicine

Table 2: Cytokine and immunoglobulin production by spleen cells in mice administered HemoHIM.

Nondiabetic Nondiabetic + HemoHIM Diabetic Diabetic + HemoHIMCytokines (ng/mL)

IL-2 1.21 ± 0.22 1.21 ± 0.19 1.22 ± 0.29 1.22 ± 0.10IFN-𝛾 6.61 ± 1.60# 6.74 ± 2.37# 2.85 ± 1.86∗ 5.01 ± 2.39IL-6 1.03 ± 0.28# 1.15 ± 0.30# 1.57 ± 0.28∗ 0.90 ± 0.31#

TNF-𝛼 0.39 ± 0.08 0.35 ± 0.15 0.53 ± 0.23 0.35 ± 0.08Immunoglobulin (ng/mL)

IgM 616.0 ± 163.2 586.4 ± 93.4# 873.5 ± 231.9 566.6 ± 290.6IgG1 5.0 ± 2.7# 5.2 ± 2.7 9.7 ± 3.6∗ 6.7 ± 1.2

The spleen cells (5 × 106 cells/mL) were treated with Con A (1𝜇g/mL) or LPS (10𝜇g/mL) for 24 hours and culture supernatants were harvested and storedat −20∘C until use. IL-2 and IFN-𝛾 were measured in the Con A-stimulated supernatant. IL-6, TNF-𝛼, IgM, and IgG1 were measured in the LPS-stimulatedsupernatant. Values are expressed as means ± S.D.∗𝑃 < 0.05 compared with nondiabetic group.

#𝑃 < 0.05 compared with diabetic group.

0

1

2

3

4

5

6

0

1

2

3

4

Nondiabetic DiabeticNondiabetic + Diabetic +HemoHIM HemoHIM

Nondiabetic DiabeticNondiabetic + Diabetic +HemoHIM HemoHIM

#

#

∗WBC

(k/𝜇

L)

Lym

phoc

yte (

K/𝜇

L)

Figure 5: ICR mice orally administrated HemoHIM (4 weeks) before being injected with STZ. Hematological change analysis was carriedout using an automatic analyzer (HEMAVET 850, USA) for white blood cells (WBCs) and lymphocytes. Values are expressed as means ± S.D.WBC: white blood cell. ∗𝑃 < 0.05 compared with nondiabetic group. #

𝑃 < 0.05 compared with diabetic group.

pancreas [27]. To investigate the infiltration of lymphocytes,pancreas was stained with H&E (see Supplement Figure1 available online at http://dx.doi.org/10.1155/2014/461685).This experiment revealed that lymphocytes infiltration wasreduced by HemoHIM in the diabetic mice.

Diabetic mice exhibit hyperglycemia in the form ofpolydipsia, polyphagia, polyuria, and body weight loss [28].The body weight of the diabetic control group decreased dueto insulin deficiency and dysfunctional energy metabolism[5, 10]. During the experimental period, the nondiabeticmiceshowed an approximately 20% body weight gain, whereasthe body weight of diabetic mice decreased by about 8%over the same time period. However, loss of body weightwas suppressed by HemoHIM. Failure of STZ-induced dia-betic animal models to gain body weight has already beenreported [29, 30]. Kidney and liver weights are higher indiabetic animals, as STZ-induced diabetic animals undergoglomerular hypertrophy [31] and triglyceride accumulation[30] in the kidney and liver, respectively. In the current study,HemoHIM attenuated hypertrophy of organs (kidney, liver,

and lung) and the thymus in STZ-induced diabetic mice[32, 33]. However, HemoHIMprotected loss of kidney weightin the STZ-induced diabeticmice but did not affect creatinine(data not shown). Dagistanli et al. [34] reported that thymicatrophy is caused by elevation of intracellular calcium levels,leading to apoptosis in STZ-induced diabetes. We also foundthat the relative thymus weight was dramatically reducedby 44% in STZ-induced diabetic mice compared to thenondiabetic group. However, HemoHIM supplementationsignificantly improved thymic atrophy by 1.8-fold comparedto the diabetic control group, which indicates thatHemoHIMmay protect the decline of immune response.

Furthermore, hyperglycemia is toxic to multiple immunecell populations, including lymphocytes [35]. Therefore, wedetermined whether or not HemoHIM affects splenocytesubpopulation in diabetic mice. Recently, immunologicalinjury was reported to be associated with DM. Avanzini etal. [36] reported that IFN-𝛾 is reduced in DM, as the per-centage of peripheral CD4+ and CD8+ cells was found to besignificantly lower in DM patients.The current study showed

Page 7: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

Evidence-Based Complementary and Alternative Medicine 7

Table 3: Plasma biomarkers in mice administered HemoHIM.

Nondiabetic Nondiabetic + HemoHIM Diabetic Diabetic + HemoHIMGOT (U/L) 55 ± 7 50 ± 9 126 ± 27∗ 73 ± 12#

GPT (U/L) 24 ± 6 21 ± 6 52 ± 23∗ 28 ± 6LDH (U/L) 368 ± 117 301 ± 31 618 ± 108∗ 489 ± 112ALP (U/L) 124 ± 11 120 ± 10 281 ± 56∗ 193 ± 20#

GOT: glutamate oxaloacetate transaminase, GPT: glutamic pyruvic transaminase, LDH: lactate dehydrogenase, ALP: alkaline phosphatase. Values are expressedas means ± S.D.∗𝑃 < 0.05 compared with nondiabetic group.

#𝑃 < 0.05 compared with diabetic group.

that spleen cells from the diabetic group produced signifi-cantly less IFN-𝛾 in response to Con A. However, Hemo-HIM recovered IFN-𝛾 production in STZ-induced diabeticmice. Further, the HemoHIM-treated diabetic group showedincreased CD4+ and CD8+ cell numbers in the spleen. Inaged mice and airway inflammation mice, HemoHIM hasbeen shown to restore the Th1/Th2 balance [9, 37]. Variousimmunoglobulins are increased in DM [38, 39]. Specifically,IgM and IgG1 were increased in the diabetic group in thepresent study. On the other hand, immunoglobulin levels ofthe HemoHIM-treated diabetic group were reduced com-pared with the diabetic control group. These data suggestthat HemoHIM treatment can overcome immunologicalinjuries in diabetic mice. Thus, HemoHIM has potentialimmunomodulatory activity in DM. Further, GOT, GPT,LDH, and ALP were significantly increased in the diabeticgroup, whereas the diabetic group treated with HemoHIMshowed restored levels. In addition, hematological resultsindicated that HemoHIM could reduce liver toxicity in STZ-induced diabetic mice.

In conclusion, HemoHIM improved the symptom ofSTZ-induced diabetic. The evidence of these characteristicsof HemoHIM includes (1) restoration from destroyed 𝛽-cells by STZ; (2) increase in blood insulin level; (3) decreasein blood glucose level by increased insulin; (4) protectionof body and organs weight from disruption by STZ; (5)inhibitition of immunological changes by STZ. On the basisof the results described herein, we suggest thatHemoHIMhasantidiabetic potential against increase in blood glucose levelsand even immune system disruption.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

This study was supported by a Grant of the TraditionalKoreanMedicine R&DProject,Ministry ofHealth&Welfare,Republic of Korea (HI12C1893), and Suncheon ResearchCenter for Natural Medicines, Republic of Korea.

References

[1] S. I. Lee, J. S. Kim, S. H. Oh, K. Y. Park, H. D. Lee, and S. D.Kim, “Antihyperglycemic effect of Fomitopsis pinicola extractsin streptozotocin-induced diabetic rats,” Journal of MedicinalFood, vol. 11, no. 3, pp. 518–524, 2008.

[2] N. S. El-Shenawy and I. M. Abdel-Nabi, “Hypoglycemic effectof cleome droserifolia ethanolic leaf extract in experimentaldiabetes, and on non-enzymatic antioxidant, glycogen, thyroidhormone and insulin levels,” Diabetologia Croatica, vol. 35, no.1, pp. 15–22, 2006.

[3] K. O. Shin, D. I. Lee, and S. D. Kim, “Diet of red ginseng-cheonggukjang improves streptozotocin-induced diabetessymptoms and oxidative stress,” Food Science and Biotechnology,vol. 17, no. 2, pp. 287–294, 2008.

[4] O. R. Bart, “The role of T-cells in the pathogenesis of type 1diabetes: from cause to cure,” Diabetologia, vol. 46, no. 3, pp.305–321, 2003.

[5] R. J. Li, S. D. Qiu, H. X. Chen, H. Tian, and H. X. Wang, “Theimmunotherapeutic effects ofAstragalus polysaccharide in type1 diabetic mice,” Biological and Pharmaceutical Bulletin, vol. 30,no. 3, pp. 470–476, 2007.

[6] R. J.Marles andN. R. Farnsworth, “Antidiabetic plants and theiractive constituents,” Phytomedicine, vol. 2, no. 2, pp. 137–189,1995.

[7] K. S. Singh, N. K. Achyut, K. G. Rajesh, J. Dolly, and W. Geera,“Assessment of antidiabetic potential of Cynodon dactylonextract in streptozotocin diabetic rats,” Journal of Ethnopharma-cology, vol. 114, no. 2, pp. 174–179, 2007.

[8] B. K. Rao, M. M. Kesavulu, R. Giri, and C. Appa Rao, “Antidia-betic and hypolipidemic effects ofMomordica cymbalaria hook.Fruit powder in alloxan-diabetic rats,” Journal of Ethnopharma-cology, vol. 67, no. 1, pp. 103–109, 1999.

[9] H. R. Park, S. K. Jo, U. Jung, and S. T. Yee, “Restoration ofthe immune functions in aged mice by supplementation witha new herbal composition, HemoHIM,” Phytotherapy Research,vol. 22, no. 1, pp. 36–42, 2008.

[10] S. H. Kim, H. J. Lee, J. S. Kim et al., “Protective effectof an herbal preparation (HemoHIM) on radiation-inducedintestinal injury in mice,” Journal of Medicinal Food, vol. 12, no.6, pp. 1353–1358, 2009.

[11] S. K. Jo, H. J. Lee, S. R. Kim et al., “Antiinflammatory activityof an herbal preparation (HemoHIM) in rats,” PhytotherapyResearch, vol. 21, no. 7, pp. 625–628, 2007.

[12] H. R. Park, E. J. Ju, S. K. Jo, U. Jung, S. H. Kim, and S. T. Yee,“Enhanced antitumor efficacy of cisplatin in combination with

Page 8: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

8 Evidence-Based Complementary and Alternative Medicine

HemoHIM in tumor-bearing mice,” BMC Cancer, vol. 9, article85, pp. 1–10, 2009.

[13] H. R. Park, E. J. Ju, S. K. Jo, U. Jung, and S. H. Kim, “HemoHIMenhances the therapeutic efficacy of ionizing radiation treat-ment in tumor-bearing mice,” Journal of Medicinal Food, vol.13, no. 1, pp. 47–53, 2010.

[14] S. K. Jo, S. H. Kim, S. T. Yee, H. R. Park, H. Oh, andM. W. Byun, “Herbal composition for improving anticanceractivity, immune response and hematopoiesis of the body, andprotecting the body from oxidative damage, and the method ofpreparing the same,” USA patent No. 6964785, 2005.

[15] S. K. Jo, S. H. Kim, S. T. Yee, H. R. Park, H. Oh, andM. W. Byun, “Herbal composition for improving anticanceractivity, immune response and hematopoiesis of the body, andprotecting the body from oxidative damage, and the method ofpreparing the same,” Europe (England, France, Germany, Italy)patent No. 1466608, 2006.

[16] S. K. Jo, U. H. Jung, H. R. Park et al., “Development of specialmedical foods and botanical drugs using HemoHIM for cancerpatients during radiation therapy,” Tech. Rep. KAERI/RR-2998/2008, Korea Atomic Energy Research Institute, 2010.

[17] H. R. Park, S. K. Jo, U. Jung, S. T. Yee, and S. H. Kim, “Protectiveeffects of HemoHIM on immune and hematopoietic systemsagainst 𝛾-irradiation,” Phytotherapy Research, vol. 28, no. 2, pp.245–251, 2014.

[18] R.Maiti, D. Jana,U. K.Das, andD.Ghosh, “Antidiabetic effect ofaqueous extract of seed of Tamarindus indica in streptozotocin-induced diabetic rats,” Journal of Ethnopharmacology, vol. 92,no. 1, pp. 85–91, 2004.

[19] J. Lee, S. T. Yee, J. J. Kim et al., “Ursolic acid ameliorates thymicatrophy and hyperglycemia in streptozotocin-nicotinamide-induced diabetic mice,” Chemico-Biological Interactions, vol.188, no. 3, pp. 635–642, 2010.

[20] S. M. Kim, J. C. Han, H. D. Park et al., “Effect of fermentedsmall soybean powder mixed with mulberry leaf on metabolicimprovement and hexokinase activity in streptozotocin-induced diabetic rats,” Nutritional Sciences, vol. 9, no. 2, pp.97–105, 2006.

[21] S. I. Jeong, D. H. Kwak, S. Lee et al., “Inhibitory effects ofCnidium officinale Makino and Tabanus fulvus Meigan on thehigh glucose-induced proliferation of glomerular mesangialcells,” Phytomedicine, vol. 12, no. 9, pp. 648–655, 2005.

[22] W. L. Li, H. C. Zheng, J. Bukuru, and N. de Kimpe, “Naturalmedicines used in the traditional Chinese medical system fortherapy of diabetesmellitus,” Journal of Ethnopharmacology, vol.92, no. 1, pp. 1–21, 2004.

[23] Y. C. Juan, W. J. Tsai, Y. L. Lin et al., “The novel anti-hyperglycemic effect of Paeoniae radix via the transcriptionalsuppression of phosphoenopyruvate carboxykinase (PEPCK),”Phytomedicine, vol. 17, no. 8-9, pp. 626–634, 2010.

[24] P. D. Whitton and D. A. Hems, “Glycogen synthesis in theperfused liver of streptozotocin diabetic rats,” BiochemicalJournal, vol. 150, no. 2, pp. 153–165, 1975.

[25] S. Golden, P. A. Wals, and F. Okajima, “Glycogen synthesis byhepatocytes from diabetic rats,” Biochemical Journal, vol. 182,no. 3, pp. 727–734, 1979.

[26] J. S. Bishop, “Inability of insulin to activate liver glycogentransferase D phosphatase in the diabetic pancreatectomizeddog,” Biochimica et Biophysica Acta, vol. 208, no. 2, pp. 208–218,1970.

[27] R. A. Rifaai, N. F. El-Tahawy, E. A. Saber, and R. Ahmed,“Effect of quercetin on the endocrine pancreas of the exper-imentally induced diabetes in male albino rats: a histologi-cal and immunohistochemical study,” Journal of Diabetes &Metabolism, vol. 3, no. 182, 2012.

[28] X. Qi, W. Chen, L. Liu, P. Yao, and B. Xie, “Effect of aSiraitia grosvenori extract containingmogrosides on the cellularimmune system of type 1 diabetes mellitus mice,” MolecularNutrition and Food Research, vol. 50, no. 8, pp. 732–738, 2006.

[29] V. Chen and C. D. Ianuzzo, “Dosage effects of streptozotocinon rat tissue enzyme activities and glycogen concentration,”Canadian Journal of Physiology and Pharmacology, vol. 60, no.10, pp. 1251–1256, 1982.

[30] J. Raju, D. Gupta, A. R. Rao, P. K. Yadava, and N. Z. Baquer,“Trigonella foenum graecum (fenugreek) seed powder improvesglucose homeostasis in alloxan diabetic rat tissues by reversingthe altered glycolytic, gluconeogenic and lipogenic enzymes,”Molecular and Cellular Biochemistry, vol. 224, no. 1-2, pp. 45–51, 2001.

[31] S. Malatiali, I. Francis, and M. Barac-Nieto, “Phlorizin preventsglomerular hyperfiltration but not hypertrophy in diabetic rats,”Experimental Diabetes Research, vol. 2008, Article ID 305403, 7pages, 2008.

[32] P. R. Nagib, J. Gameiro, L. G. Stivanin-Silva et al., “Thymicmicroenvironmental alterations in experimentally induced dia-betes,” Immunobiology, vol. 215, no. 12, pp. 971–979, 2010.

[33] E. O. Barreto, I. Riederer, A. C. Arantes et al., “Thymusinvolution in alloxan diabetes: analysis of mast cells,”Memoriasdo Instituto Oswaldo Cruz, vol. 100, no. 1, pp. 127–130, 2005.

[34] F. K. Dagistanli, B. S. Duman, andM. Ozturk, “Protective effectsof a calcium channel blocker on apoptosis in thymus of neonatalSTZ-diabetic rats,” Acta Histochemica, vol. 107, no. 3, pp. 207–214, 2005.

[35] P. C. Calder, G. Dimitriadis, and P. Newsholme, “Glucosemetabolism in lymphoid and inflammatory cells and tissues,”Current Opinion in Clinical Nutrition and Metabolic Care, vol.10, no. 4, pp. 531–540, 2007.

[36] M. A. Avanzini, L. Ciardelli, E. Lenta et al., “IFN-𝛾 lowproduction capacity in type 1 diabetes mellitus patients atonset of disease,” Experimental and Clinical Endocrinology andDiabetes, vol. 113, no. 6, pp. 313–317, 2005.

[37] J. J. Kim, H. W. Cho, H. R. Park, U. Jung, S. K. Jo, and S. T. Yee,“Preventative effect of an herbal preparation (HemoHIM) ondevelopment of airway inflammation in mice via modulationofTh1/2 cells differentiation,” PLoS ONE, vol. 8, no. 7, Article IDe68552, 2013.

[38] X. Zhang, J. Cui, M. C. Qiu, D. Q. Li, P. Zhang, and J. S. Zhang,“The effects of cyclosporine A on immunoglobulins depositionin retina of streptozotocin-induced diabetic rats,”ZhonghuaNeiKe Za Zhi, vol. 47, no. 2, pp. 125–128, 2008.

[39] J. Cui, M. C. Qiu, D. Q. Li, X. Zhang, J. S. Zhang, and P. Zhang,“The protective effects of cyclosporine A on aortic immunolog-ical injuries in STZ-induced diabetic rats,” Zhonghua Nei Ke ZaZhi, vol. 38, no. 5, pp. 440–444, 2010.

Page 9: Research Article Immunomodulatory and Antidiabetic Effects …downloads.hindawi.com/journals/ecam/2014/461685.pdf ·  · 2015-11-22Research Article Immunomodulatory and Antidiabetic

Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Disease Markers

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com